Rapid progress in genomic sequencing and proteomics has pushed the biotechnology sector to develop faster and more efficient devices for analyzing nucleic acids in biological samples. Accordingly, the biotechnology sector has directed substantial effort toward developing miniaturized microfluidic devices, often termed labs-on-a-chip, for sample analysis. Such devices may analyze samples in very small volumes of fluid, providing more economical use of reagents and samples, and in some cases dramatically speeding up assays. These devices offer the future possibility of human health assessment, genetic screening, and pathogen detection, among others, as routine, relatively low-cost procedures carried out very rapidly in a clinical setting or in the field.
Despite the potential of microfluidics, the analysis of low quantities of dilute target nucleic acids poses substantial technical problems for microfluidic devices. A typical nucleic acid analysis relies on nonselective isolation of all nucleic acids during initial sample processing. Then, a nucleic acid target(s) may be selectively amplified, generally in the presence of all of the isolated nucleic acids, to allow subsequent assay of the amplified target. However, in many cases the target is isolated in a relatively dilute form during initial sample processing and represents only a tiny fraction of the total isolated nucleic acids. For example, clinically relevant levels of human pathogens may correspond to substantially fewer than one particle or organism per microliter of human blood. Furthermore, a genetic region of interest from a low-titer pathogen or a single-copy gene may represent less than one-millionth of the total DNA isolated from a mammalian sample.
A dilute target that makes up a small fraction of the isolated nucleic acids in a sample may pose at least two problems for amplification of the target. First, because the target is dilute, a relatively large chamber, for example, up to one-hundred microliters or more, may be necessary to hold a fluid volume large enough to include a detectable number of target molecules. As a result, the need for input of a detectable number of target molecules may necessitate additional sample processing before amplification or even preclude the use of some types of microfluidic devices, particularly those that amplify and assay target nucleic acids in sub-microliter volumes. By contrast, a dilute sample in a large volume loses the benefit of microfluidic devices. Second, because the target often represents a tiny fraction of all isolated nucleic acids in the sample, amplification efficiency is reduced by the excess of non-target nucleic acids. For example, side reactions with non-target nucleic acids may slow the rate of target amplification and deplete amplification reagents, resulting at least in a decrease in signal-to-noise ratio or even a complete absence of target signal.